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Magnetic particle-isolated chemiluminescence immunoassay for detecting Tau protein (TAU)

A technology of chemiluminescence immunity and magnetic particles, which is applied in the direction of chemiluminescence/bioluminescence, analysis of materials through chemical reactions, analysis of materials, etc., can solve complicated and cumbersome operations, low automation of ELISA methods, and large pollution of RIA methods question

Inactive Publication Date: 2019-04-05
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are certain defects in these methods. The RIA method is highly polluted, the ELISA method is low in automation, and the operation is complicated and cumbersome. High performance liquid chromatography, gas chromatography, and gas chromatography and mass spectrometry are not suitable for widespread clinical testing.

Method used

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  • Magnetic particle-isolated chemiluminescence immunoassay for detecting Tau protein (TAU)
  • Magnetic particle-isolated chemiluminescence immunoassay for detecting Tau protein (TAU)
  • Magnetic particle-isolated chemiluminescence immunoassay for detecting Tau protein (TAU)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Magnetic particle labeling Tau protein (TAU) antibody

[0040] Take 20 mg of carboxyl-modified microparticle solution, settle the magnetic particles with superparamagnetic properties, uniform particle size, and carboxyl (COOH-) active groups on the surface (magnetic separation) under the action of a magnetic field for 10 minutes, remove the supernatant, and settle the magnetic particles Wash 3 times with (2-(N-morpholine)ethanesulfonic acid) MES at a molar concentration of 0.05M in the activation buffer, pH 6.0 buffer solution, 2 ml each time.

[0041] After washing, the magnetic particles were fully suspended with 1.0ml activation buffer (2-(N-morpholine) ethanesulfonic acid) MES with a molar concentration of 0.05M, pH 6.0, and then the activator 1-ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), suspension reaction at room temperature for 30 minutes, the molar concentration of EDC is 7.5mM.

[0042] Take 1.0 mg of Tau protein (TAU) anti...

Embodiment 2

[0044] Example 2: Alkaline phosphatase (ALP) labeled Tau protein (TAU) antibody

[0045] Take 1.0 mg of Tau protein (TAU) antibody, concentrate to 2.5 mg / mL, add 5 μL of activator 2-Iminothiolane hydrochloride (2-IT) solution with a concentration of 13.76 mg / mL, and react at room temperature for 15 minutes. Use Sephadex G25 gel column to desalt and collect the activated antibody.

[0046] Take 1.2 mg of alkaline phosphatase (ALP), concentrate it to 2.5 mg / mL, add 12 μL of activator Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) solution with a concentration of 6.69 mg / mL, and react at room temperature for 15 minute. Use Sephadex G25 gel column to desalt and collect activated alkaline phosphatase (ALP).

[0047] The activated Tau protein (TAU) antibody and alkaline phosphatase (ALP) were mixed according to the ratio of 1.0 mg Tau protein (TAU) antibody to 1.0 mg alkaline phosphatase (ALP), and left to react at 4°C for 18 hours.

[0048] Use Supperdex200 g...

Embodiment 3

[0049] Embodiment 3: Human Tau protein (TAU) magnetic particle separation chemiluminescent immunoassay method

[0050] (1) Immunological reaction: the 50 μl calibrator series of Example 1 (concentrations are respectively 0, 0.8, 2.5, 9.5, 25, 50 ng / ml) were respectively mixed with the quality control substance, 50 μl reagent A of Example 1, Example 1 Add 50 μl of reagent B of 2 to the reaction tube in turn, mix and incubate at 37°C for 15 minutes;

[0051] (2) Magnetic separation: settle the magnetic particles in a magnetic field, remove the supernatant, add 200-500 μl of cleaning solution, remove the magnetic field, then settle the magnetic particles in the magnetic field again, and remove the supernatant; repeat this 2-4 times, To remove unbound antibodies and impurities;

[0052] (3) Reading value: add 150 μl of luminescence substrate solution, after alkaline phosphatase (ALP) catalyzes the luminescence of the substrate, measure the relative luminescence intensity (RLU) wi...

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Abstract

The invention discloses a magnetic particle-isolated chemiluminescence immunoassay for detecting Tau protein (TAU). A kit composition comprises a calibration product, a quality control product reagentA, reagent B, concentrated cleanout fluid, and luminescent substrate solution, wherein the calibration product is Tau protein (TAU) antigen in a series of concentration and used for establishing a standard curve; the quality control product is prepared from the buffer solution containing Tau protein (TAU) antigen in a series of concentration; the reagent A is the magnetic particle marked Tau protein (TAU) antibody solution in a series of concentration; the reagent B is the alkaline phosphatase marked Tau protein (TAU) antibody solution in a certain concentration; the concentrated cleanout fluid is used for preparing the cleanout fluid; the luminescent substrate solution is the alkaline phosphatase (ALP) catalyzed luminescent substrate solution. The signal intensity and sensitivity of theimmunoreactions are greatly improved, the strong chemiluminiscence signal can be produced when the low-content substance performs the immune binding, a more accurate, precise, convenient, faster and simpler method is provided for the detection of the human body Tau protein (TAU).

Description

technical field [0001] The invention belongs to the technical field of immune detection and analysis, and provides a magnetic separation chemiluminescence immunoassay method for detecting Tau protein (TAU), which is suitable for quantitative detection of human serum Tau protein (TAU). Background technique [0002] Tau protein, also known as microtubule association protein (MAP), exists in the axons of neurons in normal brain tissue, and has the function of synthesizing and stabilizing the skeleton of neurons. The cellular function of Tau protein in the normal brain is to bind to tubulin to promote its polymerization to form microtubules; bind to the formed microtubules to maintain microtubule stability, reduce the dissociation of tubulin molecules, and induce microtubule bundles. The Tau protein molecule in normal brain contains 2-3 phosphate groups, while the Tau protein in the brain of Alzheimer's disease (AD) patients is abnormally hyperphosphorylated, each molecule of Ta...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/535G01N33/58G01N21/76
CPCG01N21/76G01N33/535G01N33/54326G01N33/581G01N33/6896G01N2800/2821
Inventor 郭健夫桂颖王义君
Owner BEIJING LEADMAN BIOCHEM
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